α-Bungarotoxin binding sites in rat hippocampus: localization in postsynaptic cells

Brain Research, 154 (1978) 167-171 © Elsevier/North-HollandBiomedicalPress

167

a-Bungarotoxin binding sites in rat hippocampus: localization in postsynaptic cells

YADIN DUDAI and MENACHEM SEGAL Department of Neurobiology and Department of lsotopes, The Weizmann Institute of Science, Rehovot (Israel)

(Accepted May 4th, 1978)

Rat brain contains specific a-bungarotoxin (a-Btx) binding sites l°-la. The pharmacological profile of these sites closely resembles that of nicotinic receptors 12, and they are differentially distributed over the brain in regions known to have a cholinoceptive nature la. However, whereas a-toxins are proven to be potent labels for nicotinic acetylcholine receptors in peripheral systems 2, it is not yet clear whether they also label functional acetylcholine nicotinic receptors in the central nervous system4. It is also not yet clear whether a-Btx binding sites in brain are presynaptic or postsynaptic. The hippocampus is one of the regions richest in specific a-Btx binding sites in rat brain la. Autoradiograms reveal specific a-[125I]Btx binding sites mainly in stratum polimorphe of the dentate gyrus and in the outer third of stratum oriens 13. These layers are also rich in acetylcholinesterase14. Most of the cholinergic input to the hippocampus comes from the septum, and destruction of the septal-hippocampal tract decreases the activities of choline-acetyltransferase and acetylcholinesterase by up to 80-90 9/00(see ref. 7). If a-Btx binding sites are present on the cholinergic presynaptic fibers, then lesions of the septohippocampal tract should also markedly decrease their level in the hippocampus. The present experiments were designed to test that possibility. We have measured changes in a-Btx binding sites level, as well as in muscarinic receptor level and acetylcholinesterase activity, in the hippocampus and hypothalamus, following fornix lesions. The hypothalamus was selected for comparison of non-specific effects of the lesion, because it contains relatively high concentration of a-Btx binding sites, and is closely associated with the hippocampus. Adult (20(0300 g) male Wistar rats were used. Rats were placed under Nembutal (45 mg/kg) anesthesia in a stereotaxic frame. A fine tipped knife (Roboz) was lowered through holes in the exposed skull, made 1.0 mm caudal to the bregma, to sever the fornix bilaterally. The rats were then kept in colony cages and allowed free access to food and water. Control, non-lesioned rats were from the same litters and were kept in the same cages as experimental animals. The rats were decapitated and the brain was rapidly removed 3-100 days later. Hippocampi and hypothalami were dissected in the cold and homogenized (100 mg/ml) in 0.32 M sucrose in a glass-Teflon homogenizer. Assays were performed on aliquots of whole homogenates.

168 a-Btx was purified from crude Bungarus multicintus venom 3 and was iodinated with 1251to an initial specific activity of 30-60 Ci/mmole by the chloramine-T method ~. [aH]Quinuclidinyl benzilate ([3H]QNB, 29.4 Ci/mmole) was purchased from New England Nuclear (Boston, Mass). [aH]Acetylcholine chloride (100-500 mCi/mmole) was from the Radiochemical Centre (Amersham). Binding of a-[lzSI]Btx was assayed as follows: aliquots of homogenate (containing up to about 0.2 mg protein) were incubated at 25 °C in 0.12 M NaCI, 2 mg/ml BSA, 0.05 M Tris. CI, pH 7.4, in total volume of 0.05 ml. Reaction was started by addition of a-[125I]Btx (final concentration 15 nM) and terminated after 100 min by diluting with 2 ml buffer, followed immediately by vacuum filtration over a Millipore EGWP 02500 filter. The filter was then washed 3 times with 2 ml portions of buffer and counted in a Packard Auto-Gamma spectrometer. Specific binding of a-[125I]Btx was defined as total binding minus the binding occurring in the presence of 0.1 m M nicotine. Each determination was carried out in a duplicate with and without nicotine. Binding of [3H]QNB was assayed as follows: aliquots of homogenate (containing up to about 0.2 mg protein) were incubated at 25 °C in 0.06 M NaCi, 0.025 M Tris • C1, pH 7.4, in total volume of0.15 ml. Reaction was started by addition of [aH]QNB (final concentration 5 nM) and terminated after 60 min by diluting with 2 ml buffer followed immediately by vacuum filtration over a glass-fiber filter (GF/C, Tamar, Israel). The filter was then washed 3 times with 2 ml portions of buffer, dried, and placed in vials containing 4 ml of 33 ~o (v/v) Triton X-100, 0.8 ~ PPO and 0.01 ~ POPOP in toluene. Vials were maintained for 12-14 h at 25 °C and counted by liquid scintillation spectrometry. Specific binding of [3H]QNB was defined as total binding minus binding occurring in the presence of 10-6 M atropine. Each determination was carried out in duplicate with and without atropine. Acetylcholinesterase was determined as described by Johnson and Russell ~, employing [3H]acetylcholine (3.3 mM) as substrate. Protein was determined according to Lowry et al. 8, using BSA as standard. Homogenates of rat hippocampus and hypothalamus contain specific a-[1351]Btx binding sites 1~. Maximum specific binding was obtained with toxin concentrations higher than 10 nM. Non-specific binding was about 40 ~ of total and was not abolished even in the presence of 1 m M nicotine, 1 m M D-tubocurarine or 10 # M non-labeled a-Btx. Half saturation of specific sites occurred at about 2 n M a-[125I]Btx. Dissociation constants (K) of various cholinergic ligands were estimated from EDs0 values (e.g., doses of ligands required to protect 50 ~ of specific toxin binding sites at saturation) EDs0 employing the relation K = • KD, where KD is the apparent dissociation [a-[lzSI]Btx] constant of a-[125I]Btx 8. The pharmacological profile of the sites is clearly nicotinic (Table I). Decamethonium, which is a potent nicotinic ligand at peripheral receptors 2 but not at ganglionic or central nervous system receptors lz, was not highly effective in protecting a-[125I]Btx binding sites. Atropine, a potent muscarinic antagonist, and oxotremorine and muscarine, specific muscarinic agonists, had little effect on toxin binding at 0.5 mM. Fornix lesions markedly decreased acetylcholinesterase activity in hippocampus,

169 TABLE I

Affinities o f various ligands for a-[1251]Btx binding sites in hippocampus homogenate Aliquots of homogenate were preincubated for 25 min with the appropriate concentrations of iigand. Reaction was started by addition of a-[laSI]Btx (15 nM) and was carried out for 120 min at 25 °C. EDso values were determined from plots of relative specific bindingvs. ligand concentration,and the dissociation constant, K, was calculated as described in the text.

Ligand

K ( M)

a-Bungarotoxin Nicotine D-Tubocurarine Acetylcholine* Gallamine Eserine Decamethonium Atropine** Oxotremorine* * D,L-Muscarine**

4 x 10-x° 1 x 10-° 2 x 10 0 1 x 10-5 3 × 10-5 2 × 10-4 2 x 10-4 > 0.5 mM > 0.5 mM > 0.5 mM

* In the presence of 1 x 10-5 M diethylfluorophosphate, which is a potent cholinesterase inhibitor but does not significantly affect toxin binding. ** Little effect at that concentration. as previously reported 7; no such effects were detected in the h y p o t h a l a m u s of lesioned animals (Table II). Muscarinic receptors level was n o t significantly altered, in agreement with Y a m a m u r a a n d SnyderlL Changes in level of specific a-[125I]Btx b i n d i n g sites were also small. Results of 3 separate experiments, covering a period of 3-100 days after lesion, are presented in Table III. F o r n i x lesion did n o t change the subcellular distribution of toxin b i n d i n g sites: in b o t h control a n d lesioned animals less t h a n 10 ~o of specific b i n d i n g r e m a i n e d in the s u p e r n a t a n t of centrifugation at 20,000 x g for 30 min, a n d in b o t h cases n o additional activity was detected by precipitating the s u p e r n a t a n t in 30 % TABLE II

Effect o f fornix lesion on the level o f nicotinic and muscarinic binding sites and on acetylcholinesterase activity in hippocampus and hypothalamus homogenates Aliquots of homogenate were assayed for a-[125I]Btx binding, [aH]QNB binding and acetylcholinesterase activity as described in the text. Results are presented as specific activities (per mg protein). Values are means 4- S.E.M. with number of animals in parentheses.

Hippocampus

Hypothalamus

Days AChE activity a-Btx QNB binding after (nmole/min/ binding (pmole/mg) lesion mg) (fmole/mg)

AChE activity a-Btx (nmole/min/ binding mg) (fmole/mg)

QNB binding (pmole/mg)

Control 10 22 80

50.6 ± 2.4 (3) 51.4 -4- 3.7 (2) 48.0 4- 0.4 (3) 53.2+4.2(3)

0.25 + 0.03 (3) 0.23 4- 0.01 (2) 0.22 4- 0.07 (3) 0.20 4- 0.06 (3)

42.3 + 1.6 (3) 6.4 -4- 0.5 (2) 6.9+0.4(3) 7.0+0.5(3)

38 -4- 2 (3) 31 4- 5 (2) 39+6(3) 33 4-2(3)

0.49 + 0.02 (3) 0.47 4- 0.04 (2) 0.55 4- 0.02 (3) 0.49 4- 0.03 (3)

36 + 5 (3) 40 4- 14 (2) 33 4-4 (3) 36 4-8 (3)

170 TABLE III Effect of fornix lesion on specific a-[1251]Btx binding and acetyleholinesterase activity in hippocampus

Aliquots of homogenate were assayed for a-[125I]Btx binding and acetylcholinesterase activity as described in the text. Results are presented as per cent specific activity relative to control, for three separate experiments. Number of animals in each experiment is given in parentheses. Days after lesion

Control 3 10 14 22 30 80 90 100

Experiment I

Experiment 11

Experiment I11

AChE activity (°o)

a-Btx binding (?/o)

AChE activity (°o)

a-Btx binding (%)

AChE activity O' (,,o)

a-Btx binding (%)

100 (2) 45 (4)

100 (2) 116 (4)

100 (3)

100 (3) --

100 (3) --

100 (3) --

--

--

--

--

--. -.

---

22 (3) -36 (3) -15 (3)

103 (3) -106 (3) -92 (3)

--

--

--

.

15

.

.

.

.

-. --

(2)

82

(2)

16 (3)

-103 (3)

17 (3)

87 (3)

20

76

(3)

(3)

s a t u r a t e d a m m o n i u m sulfate, a p r o c e d u r e t h a t precipitates soluble n i c o t i n i c receptors in p e r i p h e r a l systems 9. Specific a - B t x b i n d i n g sites in h i p p o c a m p u s are l o c a t e d in layers i n n e r v a t e d by septal afferents 13. O u r d a t a i n d i c a t e t h a t these sites are n o t localized p r e s y n a p t i c a l l y b u t o n p o s t s y n a p t i c cells. T h e o b s e r v a t i o n t h a t t o x i n - b i n d i n g sites are m e m b r a n e b o u n d a n d l o c a t e d in p o s t s y n a p t i c cells, as well as their n i c o t i n i c p h a r m a c o l o g i c a l profile a n d their differential d i s t r i b u t i o n in cholinergic r e g i o n s in t h e b r a i n la,t3 are c o n s i s t e n t with the a s s u m p t i o n t h a t they are c h o l i n e r g i c receptors. H o w e v e r , their precise p h y s i o l o g i c a l n a t u r e has yet to be d e t e r m i n e d . This w o r k was s u p p o r t e d b y g r a n t s f r o m the U n i t e d S t a t e s - I s r a e l B i n a t i o n a l Science F o u n d a t i o n , J e r u s a l e m (to Y . D . a n d M.S.). Y . D . is a n i n c u m b e n t o f the B a r e c h a F o u n d a t i o n C a r e e r D e v e l o p m e n t C h a i r a n d M.S. a n i n c u m b e n t o f t h e W o r m s e r C a r e e r Development Chair.

1 Berg, D. K., Kelly, R. B., Sargent, P. B., Williamson, P. and Hall, Z. W., Binding of a-bungarotoxin t o acetylcholine receptors in mammalian muscle, Proc. nat. A cad. Sci. ( Wash. ), 69 (1972) 147-151. 2 Changeux, J. P., The cholinergic receptor protein from fish electric organ. In L. L. Iversen, S. D. Iversen and S. H. Snyder (Eds.), Handbook ofPsychopharmacology, Vol. 6, Plenum Press, New York, 1976, pp. 235-301. 3 Changeux, J. P. ,Kasai, M. and Lee, C. Y.,Use of a snake venom toxin to characterize the cholinergi ~ receptor protein, Proc. nat. Acad. Sci. (Wash.), 67 (1970) 1241-1247. 4 Duggan, A. W., Hall, J. G. and Lee, C. Y., Alpha-bungarotoxin, cobra neurotoxin and excitation of Renshaw cells by acetylcholine, Brain Research, 107 (1976) 166-170. 5 Johnson, C. D. and Russell, R. L., A rapid, simple radiometric assay for cholinesterase suitable for multiple determinations, Analyt. Biochem., 64 0975) 229-238. 6 Levitzki, A., Sevilia, N., Atlas, D. a n d Steer, M. L., Ligand specificity and characteristics of the/Jadrenergic receptor in turkey erythrocyte plasma membranes, J. molec. Biol., 97 (1975) 35-53.

171 7 Lewis, P. K., Shute, C. C. D. and Silver, A., Confirmation from choline acetylase analysis of a massive cholinergic innervation to the rat hippocampus, J. Physiol. (Lond.), 191 (1967) 215-224. 8 Lowry, O. H., Rosebrough, N. J., Farr, A. L. and Randall, R. J., Protein measurement with the Folin phenol reagent, J. biol. Chem., 193 (1951) 265-275. 9 Meunier, J.-C., Olsen, R. W., Menez, A., Fromageot, P., Boquet, P. and Changeux, J. P., Some physical properties of the cholinergic receptor protein from Electrophorus electricus revealed by a tritiated a-toxin from Naja nigricollis venom, Biochemistry, 11 (1972) 1200-1210. 10 Polz-Tejera, G., Schmidt, J. and Karten, H. J., Autoradiographic localization of a-bungarotoxinbinding sites in the central nervous system, Nature (Lond.), 258 (1975) 349-351. 11 Salvaterra, P. M., Mahler, H. R. and Moore, W. J., Subcellular and regional distribution of 1251labelled a-bungarotoxin binding in rat brain and its relationship to acetylcholinesterase and choline acetyl transferase, J. bioL Chem., 250 (1975) 6469-6475. 12 Schmidt, J., Drug binding properties of an a-bungarotoxin-bindingcomponent from rat brain, Molec. PharmacoL, 13 (1977) 283-290. 13 Segal, M., Dudai, Y. and Amsterdam, A., Distribution of an a-bungarotoxin-bindingcholinergic nicotinic receptor in rat brain, Brain Research, 148 (1978) 105-119. 14 Storm-Mathisen, J., Quantitative histochemistry of acetylcholinesterase in rat hippocampal region correlated to histochemical staining, J. Neurochem., 17 (1970) 739-750. 15 Yamamura, H. I. and Snyder, S. H., Postsynaptic localization of muscarinic cholinergic receptor binding in rat hippocampus, Brain Research, 78 (1974) 320-326.